Steroid production



Dec. 10, 1957 J A. CAMPBELL ETAL STEROID PRODUCTION Filed May 5, 1954 llINVENTORS J A.CAMPBELL, DOUGLAS A. SHEPHERD ARNOLD s. UTT ""0 BRYONAJOHNSON ATTORNEY United htates Patent sTEnom rnonoorrors ApplicationMay 3, 1954, Serial No. 427,078-

32 Claims. (Cl. 260-3972) This invention relates to a novel process forthe sepa ration and thepurification of certain steroids, moreparticularly, to the separation and purification of stigmasterol andsitosterol and to a novel procedure for their separation and novelcompounds produced therein.

Itis an object of the present invention to provide a novel process forthe separation and purification ofstigmasterol and/or sitosterol.Another object is the provision of a process for the isolation of astigmasteryl carbamate from a mixture with sitosteryl carbamate. Stillanother object is the provision of a process for the isolation of asitosteryl carbamate from a mixture witha stigmasteryl carbamate. Stillanother object is the provision of a process for the separation ofstigmasteryl ot-naphthylcarbamate from a mixture with sitosterylOtnaphthylcarbamate in high purity. A further object is the provision ofa novel procedure for the separation-of stigmasterylct-naphthylcarbamate in high yield and in high purity from a mixturewith sitosteryl a-naphthylcarbamate. Yet another object is the provisionof the novel soysteryl a-naphthylcarbamate, stigmasteryltat-naphthylcarbamate, and sitosteryl m-naphthylcarbarnate. Otherobjects will be apparent to those skilled in the art to which thisinvention pertains.

According to the process of the present invention, a mixture of astigmasteryl carbamate and the corresponding sitosteryl carbamate isparticularly dissolved in an organic solvent and the portion enrichedwith the desired carbamate is separated and once more partiallydissolved in an organic solvent, the separation step of the enrichedportion and partial solutionstep each being repeated at least once more.The starting mixture of stigmasteryl carbamateand a correspondingsitosteryl carbamate is ordinarily obtained by the conversion of amixture of stigmasterol and at least one of the sitosterolsto one oftheircorresponding carbamates.

Reference is made to the accompanying drawings which illustrate variousfractionation techniques available forthe separation or purification ofone or more of the components of the mixture [see Morton, LaboratoryTechnique in- Organic Chemistry, McGraw-Hill Book Company, Inc. (1938),chapter VII]. In the drawings, M

' refers-to a portion of the starting mixture of a stigmasterylcarbamate and the corresponding carbamate of sitosterol, S refers to avolume of solvent, E refers to a solution, R refers'to theundissolvedportion ofthe startingmixtureand F refers to a fraction'ofthe originalmixture.-

In the drawings:

Fig. lrepresents asimple repeated fractionation with solvent in whichthe undissolved portion is re-treated with solvent.-

Fig. Z-represents asirnple repeated fractionation with solventin whichthedissolved portion is freed of solvent and then re-treated withsolvent.

Fig. 3 represents a triangular fractionation wherein both theundissolved and dissolved portions are-further 0 4378 (1906), developeda technique of separating stigfractionated; Inr. this-= procedure,afterfractionating the.

2,8 16,1 19 Patented Dec. 10 1957 mixture with solvent into a portiondissolved in the mother liquor and an undissolved portion, theundissolved portion is again fractionated with more solvent intodissolved and undissolved portions, and the mother. liquor consistingthe dissolved portion is concentrated, diluted with other solvent, orcooled, to precipitate a portion of the dissolved solids which is thencombined with the-mother liquor obtained from the second fractionationof the undissolved portion. This procedure is then repeated untilmaterial of desired purity is obtained.

Fig.. 4 represents a batchwise countercurrent type'of fractionationwherein a portion of the starting mixture is serially treated withsolvent to give several mother liquors which are used in the orderobtained to serially fractionate a second portion of the mixture, addingadditional solvent to the mother liquors to-maintain a conliquors areemployed as fractionating solvents with fresh solvent being employedafter the mother liquors.

Stigmasterol and sitosterol commonly occur together in various naturalsources, e. g., soybeans, sugar cane oil, calabar and coffee bean, etc.When the sterols are'recoveredfrom these natural sources, thestigmasterol and sitosterol are recovered as a mixture. Sincestigmasterol is ordinarily a minor constituent of these sterol fractionson a weight basis, the crude sterols when obtained'from the usualsources require an extensive pun'ficationbefore stigmasterol ofreasonable purity is obtained. For most purposes, the separation ofstigmasterol and sitosterol'is highly advantageous, because thesynthetic techniques of converting stigmasterol into hormones orhormone-like compounds, e. g., progesterone, androsterone, ordehydroepiandrosterone, etc, are quite dissimilar from those employingsitosterol as the starting steroid.

The processes heretofore available for the separation of stigmasterolfrom the sitosterols have been complicated and relatively ineffective.Windaus and Hauth, Ben, 39,

masterol from the sitosterols by preparing their acetates, brominatingthe double bonds of the steryl acetates, separating the lesser solublestigmasteryl acetate tetrabro nide which is then debrominated andsaponified to regenerate stigmasterol. The process is disadvantageous inits multiplicity of steps and its failure to recover much'of thestigmasterol by thisprocess [Neu and Eh'rbacher, Arch. Pharm., 283, 2271950)].

It has now been found in accordance with this invention thatstigmasterol or sitosterol, or both, as a mixture of one of theircarbamates, are easily separated or purified by contacting the mixedsteryl carbamates with an organic solvent according to the proceduredescribed hereinafter. If the available steroid mixture consists ofstigmasterol and sitosterol, e. g. soysterols, the sterol mixture isconverted to a mixture of one of their corresponding carbamates prior tothe separation step. This can be accomplished by any of several methods,prototypes for which may be found in the citations here listed. Forexample, the stigmasterol and sitosterol can be reacted witha-naphthylisocyanate [Neuberg and Kansky, Biochem. Z., 277, 345 (1910)].Another technique is to react a mixture of stigmasterol and sitosterolwith phosgene to produce their chloroformate esters which are thenreacted with u-naphthylamine to produce the anaphthylcarbamate esters ofstigmasterol andsitosterol lwieland, Honold and Pascual-Vila, Z. Phys.Chem., 130, 335 (1923)]. Other techniques involve the reaction of thestigmasterol-sitosterol mixture with a-naphthylcarbamyl' chloride[Lesser, Kranepul and Gad, Ber., 58, 2122 (1925)]; Herzog, Ber., 40,1831 (1907)] or with ot-naphthoic acid azide [Sah et al., Science-Repts. Natl,

Tsing Hua Univ., (A) 3, 109 (1935)]. See also Cheronis and Entrikin,Semimicro Quantitative Organic Analysis, Crowell Co., New York, page 227(1947); Org. Syn., Collective Vol. I, 140 (1941); and Kraft, I. Am.Chem. Soc., 70, 3570 (1948). Among these methods, theanaphthylisocyanate method is the most convenient and is usuallypreferred.

stigmasteryl a-naphthylcarbamate and sitosteryl anaphthylcarbamate canbe prepared in almost any inert organic diluent in which the reactantsare somewhat soluble and, at least in the case of theot-naphthylisocyanate method, no solvent need be employed. Dependingsomewhat upon the particular reagent employed to produce the steryla-naphthylcarbamates, reaction temperatures between about zero degreescentigrade and the boiling point of the reaction mixture can beemployed. Reactions employing higher temperatures than about 170 degreescentigrade are usually complicated somewhat by accompanying sidereactions. Reactions employing temperatures substantially below roomtemperature usually require prolonged reaction times and the reaction issometimes complicated by the precipitation of the reactants from thereaction mixture. The solvent employed in the preparation of thea-naphthylcarbamates can sometimes advantageously be employed as theseparation solvent, thereby eliminating any transfer or handling of thereaction mixture.

In carrying out the separation or purification step of the presentinvention, a mixture consisting essentially of one of the carbamates ofstigmasterol and of at least one of the sitosterols is fractionated intoa dissolved portion and an undissolved portion by contacting the mixturewith organic solvent. The portion which is enriched with the desiredcarbamate which is to be separated or purified is then separated fromthe organic solvent and the portion enriched with the undesiredcarbamate. The portion enriched with the desired carbamate is thenfractionated into a dissolved and an undissolved portion by contactingthe enriched portion with organic solvent. The steps of separating theportion enriched with the desired carbamate from the organic solvent andthe portion enriched with the undesired carbamate and fractionating theportion enriched with the desired carbamate into a dissolved portion andan undissolved portion by contacting the enriched portion with anorganic solvent are repeated at least once more.

The fractionation of the mixture of steryl carbamates into anundissolved portion and a dissolved portion can be achieved bycrystallization, i. e., dissolving all of the solids and thenprecipitating a portion of the dissolved solids, or by leaching(extracting), i. e., by dissolving only a portion of the total solids.

Of the procedures for separating the steryl carbamate mixture into adissolved portion and an undissolved portion, leaching of the mixture ispreferred. The method is uncomplicated, usually employing roomtemperature, and requires neither distillation apparatus nor heating andcooling.

Solvents which can be employed in the separation step include thealiphatic, alicyclic, and aromatic hydrocarbons, and like neutralnon-polar solvents; esters and ketones, and like basic polar solvents;halogenated arematic and aliphatic hydrocarbons, and like neutral polarsolvents; and other organic liquids in which at least one of the sterylcarbamates is significantly soluble. The hydrocarbons and chlorinatedhydrocarbons, both aromatic and aliphatic, are ordinarily the mostuseful, with chlorobenzene producing outstandingly desirable results ina countercurrent leaching technique such as described in the exampleshereinafter.

While a wide variety of organic solvents can be used to effect theseparation of the mixture of steryl carbamates in accordance with theprocess of the invention, the more efiicient fractionations employ thosesolvents combining 4 Some solvents, for example, while exhibiting a highdifferential solubility, that is, a high ratio of solubility of one ofthe steryl carbamates of the mixture to the solubility of the othersteryl carbamate of the mixture, have such low absolute solubility thatexcessively large volumes of solvent are required to effect theseparation, thus reducing the efiiciency of the operation. Highdifferential solubility and high absolute solubility, however, are notthe only factors which determine the most efiicient solvents. Forexample, some solvents, while exhibiting a very high differentialsolubility, appear to be less efficient than other solvents with lowerdifferential solubility ratios. As shown in Table II below, whencomparing the solubilities of stigmasteryl u-naphthylcarbamate andsitosteryl a-naphthylcarbamate in various solvents, cyclohexane has ahigher difierential solubility ratio than chlorobenzene. However, whenfractionating a mixture of stigmasteryl a-naphthylcarbamate andsitosteryl u-naphthylcarbamates, chlorobenzene is the solvent of choiceover cyclohexane because, although having a lower solubility ratio,chlorobenzene is a more efficient solvent in the separation and willseparate stigmasteryl m-naphthylcarbamate of higher purity and in higheryield than cyclohexane. Moreover, the higher absolute solubility ofchlorobenzene for the two carbamates reduces the total volume of solventnecessary and therefore makes the process more efiicient on a largescale. A higher recovery of stigmasteryl a-naphthylcarbamate of somewhatlower purity can sometimes be obtained with solvents other thanchlorobenzene, however.

Ordinarily, the stigmasteryl carbamate derivative will be found to bemore insoluble than the corresponding sitosteryl carbamate derivative.Thus the portion of the mixture which is undissolved by the solventusually is the portion which is enriched with the stigmasterylderivative.

The fractionations are advantageously carried out at room temperature,i. e., leaching the mixture with solvent at room temperature andconducting the separation at room temperature or dissolving the mixtureat a temperature above room temperature and thereafter precipitating aportion of the dissolved material at room temperature. Not only is roomtemperature preferred for convenience, but the solvents are usuallysomewhat more efficient at room temperature than they are at moreelevated temperatures. For the most part, solvents appear to lose someof their selectivity at more elevated temperatures and therefore usuallyhave lower purification efiiciencies.

Of the variables which influence the efficiency of the process of theinvention, the most important is the particular carbamate mixture to beseparated. Whereas the process of this invention broadly is applicableto the separation of any mixture consisting essentially of a carbamateof stigmasterol and the corresponding carbamate of at least one of thesitosterols, the preferred and more useful starting carbamate mixturesconsist of a stigmasteryl arylcarbamate and the correspondingarylcarbamate of sitosterol. Examples of these include thephenylcarbamate, a-naphthylcarbamate, o-biphenylcarbamate,p-biphenylcarbamate, o-tolylcarbamate, m-nitrophenylcarbamate. Of thearylcarbamates, the phenylcarbamate and the a-naphthylcarbamate of thesoysterols are preferred, especially the a-naphthylcarbamate mixturewhich is the most readily separated by the process of this invention andaffords separated stigmasteryl a-naphthylcarbamate and/ or sitosteryla-naphthylcarbamate of high purity. Another eflicient carbamate mixtureis soysteryl phenylcarbamate. Tables I and 11 below give thesolubilities of stigmasteryl and sitosteryl phenylcarbamates in varioussolvents and the ratio of their two solubilities and the solubilities ofstigmasteryl u-naphthylcarbamate and sitosteryl tx-naphtylcarbamates andtheir solubility ratios in various common organic solvents.

In determining the solubilities of stigmasteryl and ferred toataredWeighing dish'and evaporated. The residue was'driedfat fiftydegrees 'ce'ritigradeat reduced pressure. The Weightdf' residueobtainedfrorn the measured "TABLE gr gsorosmrrns on STERYL PHENYL-CARBAMA IES- IN VARIOUS SOLVENTS Solubility g./100 ml. solventSolvnt;'*27 *0. a 1 Reno sitosteryl Stigmasteryl Ph'e'nyl- Phenyl- Icarbamate carbamate ,c clohexane 2.-2 -;4 5.5 b enzene.-- li la tat Y ety Ce e 6.4. 1:4: 358 9.6 2.0 4.8 *as' 1:5 as 15. E 1 5.1 3.0 0.9 0.2.4.5 16.6 QQ'O TABLE II.SOLUBILITIES 0F STERYL a-NAPH- THYLCARBAMATES INVARIOUS SOLVENTS Solubility; g./100 m1.

solvent Solvent 25 0. 1 p Ratio 'Sitoste'ryl Stigmasteryla-Naphthyla-Naphthylcarbamate carbamate benzene 13. 8 0. 4 32 benzene(80 0.; per 100 gr 501) 50 3. 7 14 ethyl acetate 2. 5 0. 4 6. 2isopropyl alcohol- 0.3 -0. 01 30 efiorogenzene 1 3 2 6 "1.06.. 19. 9 0.7 29 en ne er 22s- .r. 5... ,7. 7.. 8:8 0. 3 2. 9 0.9 0.1 9 4. 3 0.1 43l8 methoxyethanol- 0. 4 cyclohexane 1. 5 0. 04 37 cyclohexane (80 0.;per 100 gr. 5121.). 31. 6. 1.1 29 methyl ethyl ketono 3. 5 0. 16 22methyl ethyl ketone (80 0.; per

100 gr. sol.) 38. 1 1. 6 24 hexane 2069; $8) hexane (60 0: per 100 gr v4 1:1 cyolohexane-CHgClz 26.3 1. 1 24 It is readilyapparent from TablesI and II that one of the factors which makes the u-naphthylcarbamatemlxture a mixture which is separated more efiiciently, is the highsolubility ratio of sitosteryl a-naphthylcarbamate to sagmastrylor-naphthylcarbamate in organic solvents.

During a separation with organic solvent, the mixture is preferablystirred to ensure complete contactof the solvent with all the solidparticles. The particle size of the solid should be small enough toprevent significant amounts of the solid from being shielded from thesolvent by the solid on the surface of the particles. Material of 100mesh size or smaller particlesize is sufficiently small to achieveefiicient separation, although material containing much larger particlesis operable-starting material.

To overcome the low yield of pureseparated carbamate which is obtainedfrom a repeated crystallization or-leach- 7 ing procedure such as isshown-in Figures 1 and let the drawings, othermore efficient techniquescan he em- "ployed. For example, a'trian'gular crystallization of;-the

typeshown in Figure 3 'of the drawings and illustrated in Examples '4 to13 hereinafter recovers all of the starting material as severalfractions(if-varying composition.

'A'r'nore preferred technique of recovering more of'the availabledesired carbamate in the original mixture involves a countercurrent typeof fractionation. Figure 4 "of 'the'drawings illustrates abatch-wisecountercurrentgtype procedure which employs the mother liquors from oneseparation as the solvent in further separations, thereby reducing theloss'of desired carbamate in the mother liquors. A more efficientbatch-wise countercurrent separation technique is illustrated in Figure5 of the drawings wherein only a portion of the mother liquors are usedalong with fresh solvent. The proportion of mother liquors which will bereused in a procedure of this type and the portion of fresh solventwhich is used will vary some with the particular carbamate and solventemployed. An example of this type of separation may be found in Example19 hereinafter.

Ordinarily, a leaching separation'igives higher yields of more highlypurified'separated carbamate and for this reason is usually thepreferred procedure.

A preferred leaching technique involves the use of a mixture ofstigmasteryl and sitosteryl a-naphthylcarbamates. When using thisstarting mixture, the mixture is leached to leave an undissolved portionenriched with stigmasteryl a-naphthylcarbamate which is separated andleached again with solvent. The undissolved portion is then separatedand leached at least once more. If this procedure is continued,substantially pure stigmasteryl oznaphthylcarbamate ultimately will beobtained as the remaining undissolved solid, with substantially puresitosteryl u-naphthylcarbamate being obtained in .the first motherliquor. Preferably the leaching is conducted at about room temperature,i. e.,' between about'twenty and about thirty degrees centigrade withthe preferred leaching solvent being chlorobenzene.

A refinement of the above described leaching procedure involves at leasttwo leaching operations, each of'wliich uses a volume of between aboutone and about three-niilliliters of chlorobenzene per gram of originalmiXtuIreto be fractionated, with the chlorobenzene containing sitosteryla-naphthylcarbamate and s'tigmasteryl wnaphthyl'carbamate, the amount ofsitosteryl u-naphtl'iylcarbamate, decreasing and the proportion ofstigmaste'ryl a-naphthylcarbamate to sitosteryl oc-naphthylca'rbai'nateincreasing with successive portions ofthe chlorobenzene, followed by atleast one more leaching operation using chlorobenzene of a volumebetween about oneand about three milliliters per gram of the originalmixture. When employing this procedure, high purity stigmasteryl 0t--naphthylcarbamate is obtained. This leaching procedure is then repeatedwith a second portion of steryl a-naphthylcarbamate, employing at leastall except the first two mother liquors, in the order obtained, "from'the fractionation of the first steryl u-naphthylcarbamate portion,followed, as with the first portion, by a portion of chlorobenzene. Whenthis procedure is repeated sevral times, a batch-wise countercurrentfractionation of the };type illustrated by Example 19 hereinafter isperformed. -This refinement results in the isolation of stigmasteryltat-naphthylcarbarnate of high purity and yield. Hereal'so the preferredfractionation temperature is room temperature and the leachingoperations involving chlorobenzenecontain-ing dissolved steryla-naphthylcarbama-te are preferably performed at least four times andthe final leaching operation with chlorobenzene is preferably performedat least twice. The preferred volume of solvent under these conditionsfor each leaching operation is'about two milliliters per gram of theoriginal mixture.

The stigmasteryl carbarn'ate or sitosteryl car'harnate separated orpurified according to the .processofthis invention can be assayed byhydrolyzing the isolated carbamate to produce the free sterol which isthen titrated with a standardized bromine solution according toprocedures known in the art. Pure stigmasterol will titrate two moles ofbromine per mole of sterol and pure 'y-Sit0S- teryl, for example, willtitrate one mole of bromine per mole of steroid.

stigmasteryl carbamates and sitosteryl carbamates, which are soysterylcarbamates, are represented by the following structural formulae:

wherein R in each formula is an amino group, e. g., an unsubstituted,mono or di-substituted aromatic, aliphatic, carbocyclic or heterocyclicamino group. Representative soysteryl carbamates include those wherein Ris NH methylamino, n-butylamino, octylamino, dodecylamino, phenylamino,benzylamino, para-biphenylamino, ot-naphthylamino, cyclohexylamino,clyclopentylamino, pyrrolidyl, piperidyl, morpholino, 2-pyridylamin0,dimethylamino, diphenylamino, and diethylamino. The term soysterylcarbamate defines a mixture of steryl carbamates obtained from theesterification of the soybean sterols to produce a mixture of one oftheir carbamates. Soysterols consist predominantly of about ten to about25 percent stigmasterol and about 75 to ninety percent 8 Hirshberg,Biochem. Z., 27, 345 (1910)] and of ergosterol [Windaus, Nachr. Ges.Wiss. Gottingen, Math. Phys. Klass, 202 (1928); see Elsevier,Encyclopedia of Organic Chemistry, Series III, 14, 75 (1940)] are knownin the art. The phenyl carbamate of sitosterol is also known [Heiduschkaand Gloth, Arch. Pharm., 253, 415 (1915)].

When following the separation procedure of this invention, eitherstigmasterol or sitosterol can be separated from a mixture of the two,e. g., soysterols, with only two reactions involved, viz., theesterification of the sterol mixture, to give the desired carbamatemixture and the hydrolysis of the separated stigmasteryl carbamateand/or sitosteryl carbamate. This process of separation is much shorter,compared with the four reactions of Windaus and the five reactions of U.S. Patent 2,520,143, and moreover eliminates the use of the expensiveand somewhat dangerous and corrosive bromine. Thus with the techniqueswhich have been developed for the separation of stigmasteryl carbamate,outstandingly high recoveries of the stigmasterol present in theoriginal sterol mixture is obtained as evidenced by the exampleshereinafter. For example, whereas the Windaus et al. method results in arecovery of no more than about 35 to percent of the stigmasterol presentin the average mixture of soysterols, about 65 to 85 percent of thestigmasterol present can be obtained from the same soysterols using thepreferred separation procedures described hereinafter.

The process of the invention has the advantage that a mixture of acarbamate of stigmasterol and the corresponding carbamate of at leastone of the sitosterols is more readily separable than is a mixture ofthe corresponding free sterols. Moreover, the preferred carbamatemixtures employed as starting material in the process of this inventionpermit the isolation therefrom of the carbamateof stigmasterol and/ orsitosterol in high purity with surprising ease. Further reactions,designed to produce derivatives of stigmasterol and sitosterol which canbe separated, such as brominations of the acetates of stigmasterol andsitosterol in mixture, are unnecessary to achieve separation orpurification of one of the components of a mixture consistingessentially of one of the carbamates of stigmasterol and at least one ofthe sitosterols.

Included in the stigmasteryl carbamates, which can be isolated andpurified according to the process of this invention, are those which arerepresented by Formula I above wherein R has the values given in TableIII below. These carbamates, when purified to a high degree and thenfinally crystallized from the solvents shown, have sitosterol, the termsitosterol defining the mixture of the physical constants shown in TableIII.

Analysis [71],, M. P.. C. carbamate R= Formula M. W. Calcd. Found CHCls(Kofler) Crystallizing Solvent degrees a-Naphthylammo. CmHssNOz 581. 8482. 56 9. 53 82. 21 9. 38 2 213-217 Cyfillohezane-ltlethylene c on c.

Phenylamino 03511531902 531. 24 81.39 10.05 81.70 10. 13 -38 194. 5-196.0 Cyclohexanc. p-Nitrophenylamino... O36H52N204 576. 80 74. 96 9. 09 74.94 9. 23 -34 228-230 Ethyl acetate.

o-Biphenylamino 0421-1571902 607. 89 82. 98 9. 45 83. 18 9. 3O 18194-196 Cyclohexanc.

p-Tolylamino- C37I155NO2 545. 82 81. 41 10. 15 81. 71 10. 41 36 194-196.5 Skellysolve C. oolylamino C31H N02 515. 82 81. 41 10. 15 81.75 10. 24-35 166-168. 5 Acetone. Octadecylammo 04311 910: 702. 17 81. 24 12. 2081.53 11. 89 27 104-105 Ethyl acetate. Dlethylamino CHH NOZ 511. 81 79.78 ll. 23 80.03 11. 09 -34 146-148. 5 Acetone. n-Butylamiuo Os4I-1s7NOz511. 81 79. 7 S 11. 23 80.02 11. 36 -38 139-141 Acetone-methanol. 2GZOI'IQNOz 455. 79. 06 10.84 79.07 11.12 48 225-229 Ethyl acetate.methylamino C HHNO 469. 78 79. 26 10. 94 79. 51 10. 64 -45 205-210 D0.dehydroabietylamino CLOIITIN O2 724. 13 82. 93 10. 72 83. 61 10. 83 +1154-159 Ethyl acetate-Acetone.

side-chain saturated sterols, e. g., a-sitosterol, ca -81108161701, LK-SitOStEI'OI, or -sitosterol, fi-sitosteroi and 7-Sit0st610l, the latterbeing the predominant soysterol [see Fieser and Fieser, Natural ProductsRelated to Phenanthrene, 159, 285 (1949); Markley, Soybeans and SoybeanProducts,

'Interscience Publishers, 199, 836 (1950)].

The a-naphthylcarbamate of cholesterol [Neuberg and -and-rosterone hasbeen well established [Heyl-and Herr,

-.J.;Am. Chem. Soc.,-7-2, 2617 (1950);-Fieser and-Fieser, iilflaturalProducts Related to Phenanthrenef Third .Edition, 385 to 387 .(l949)].These useful compounds have-also been used to prepare -other usefulsteroids, n.e...,g.,\dihydroisoandrosterone, cortisone, hydrocortisone,Itestoste'rone, 1'7u,2l-dihydroxyprogesterone, ZL-hydroxy-..=progesteron e, andmany others.

The hydrolysis of the separated stigmasteryl carbamate.br-sitosterylcarbamatecan beflcarried out-aceording-to usual proceduresfor effecting hydrolysis of esters. A particularly satisfactory method,however, involves the se of-a single phase hydrolysis systemfe. 'g.,-a-solution -th'e" carbamatein *a somewhat water-soluble organicsolvent, e. g. ethylene glycol,-"t'rimethylene"glycol, pro-"pyleneglycol; monoethers or'esters of glycols, especially-ethyleneiglycol monomethyl ether (Methyl Cellosolve), tniethanoh'ethanol,etc.,- containing water'and a hydrolyzing agent, e. g.,hydrochloric acid, sulfuric acid, sodium hydroxide, potassium hydroxide,*especiallysodium "hydroxide or 'potassium hydroxide which produce analmost quantitative hydrolysis. Although -lower temperatures areoperable to a lesser extent, the usual reaction temperature is aboutIOOdegrees centigrade or higher, e. g., the boiling point ofthereaction'mixture.

The reaction time required to obtain satisfactory hydrolysis dependssomewhat upon the reaction temperature, the hydroly zing agent, and thecarbamate to be hydrolyzed, about two hours being usually sufficientwhen potassium or sodium hydroxide is employed and thereaction mixtureis maintained at about- 100 degrees centigrade.

When the hydrolysis is substantially complete, the product canbeextracted from the reaction'mixturewith benzene, toluene, methylenechloride, or other solvent in which 'the thus-obtained*stigmasterol or-sitostero1 is significantly solubleg either before or after theneutralization of any-acidic or alkaline materials in "the "reaetionmixture. A suitable-method involves dilution of the reaction mixturewith fwater-and-then extracting ljwith toluene. The amineproduced in'thereaction mixtu-re "will alsobe extracted if the reaction mixture isalkaline and -thiscan-be removed from the solvent extract by washingaqueous acid. When the hydrolysis is performed 'nder acidic conditions,the amine'r' emains in the aqueous 'yeras an'a'cid addition'saltWhichcanbe neutralized andtheamine recovered by conventionalmethods."Distilling the solvent from the extractdeavesa[distillation 3 resi"dueconsisting essentially of'the steroh i.e., stigmasterol f "b'r a sitosterol. Alternatively, the reaction mixture can be dilut'e dwith waterto precipitate the' hydrolyzed "sterol 1 and the-'ste'rblfiltered fromthe reaction mixture. Washihg o'rslurryingthe resulting precipitate withfdilute aqueous aeid"wil1 remove the aminewhich precipitates with the"stigmasterol if the rea'citiontnixture is alkaline.

The purityof the thus-produced stigmasterolwill depend up'on theefficiency of the separation technique and 'upon 1 the reactionconditions for the formation 'and' hydrolysis "of the' 'stigmasterylcarbamate. When optimum conditibnsare-emp'loyed, the stigmasterol will'be'obtained in boutiiinetyp'erc'ent or better'purity andwillnotreqnirefurtheflpiirifieation' for most purposes. A simple crystallization in"the usual 1 manner will purify somewhat the l'l-ydrolyzed stigmasterol.

The yield of hydrolyzed stigmasterohcalculated upon sotope: percentpurity, "can be achieved-when optimum conditions for the preparation,separation and hydrolysis 'steps are employed.

PREPARATIONr-STERYL CHLOROFORMATE boiled to remove any water and thenfiltered through diat'omaceous earth. The filtrate was diluted with oneliter of benzene and'cooled to room temperature. 'Phosgenewas thenpassed into the solution intermittently for :grams ofsterylchloroformate dissolved in 300 milliliters of benzene wasshakenvigorously in a separatory funnel,

the benzene layer separated, washed with water, andhydrochloric acid andfinally with water. The benzene --solution was filtered and'distilled todryness to yield 20.3

.. grams of steryl methylcarbamate.

B. Steryl dhydroabietylcarbamate.Fo1lowing the procedure described inPreparation A, but substituting an equimolar amount ofdehydroabietylamine forthe aqueous methyl amine, there was producedsteryl dehydroa-bietylcarbamate in 97.5 percent yield.

"temperature and -was maintained at that temperature while passingphos'gene intothe solution for one hour., The

' methylene chloridewas distilled'andthe residue freed of solventbyheating-at reduced, pressure. The distillation "residuewas-dissolved-in300 millilitersof dry methy lene chloride andammoniagas was passed intothe solution for about 'twenty minutes. The reaction mixturewasthenconcentrated to about 200 milliliters'by distillation at 'a'tmospheric.pressure. One hundred milliliters of I. fresh 'r'nethylene chloride wasadded-and the mixture, which now contained 'aprecipitate, was filtered.The filtrate was concentrated until crystals began to form and 800milli- I liters of acetone was then added. The solution -wasdistilledlto'a volume of about 500 milliliters and crystals once morebegan to form. The mixture was cooled to about five degrees -c'entigradeand the thus-precipitated crystals were b'ollect'e'd "by filtration andwashed with cold acetone. Therewasthus obtained 16.5 grams, a:yield of75 percent of thetheoreticahof soysteryl carbamate melting at 200 to2'l2 degrees centigrade (Kofier) D. Soysteryl-n'-butylcarbamate.-Following the procedure described 'in Preparation C,usingtwenty grams of soysterols but substituting lllgrams ofn-butylamine for the ammonia, there was obtained soysteryln-butylcarbamate.

- E. soysteryl t-naphthylcarbamata one hundred grams -of.s'oysterolsconsisting of eighteenpercentstigmasterol were dissolved in one liter ofhot benzene a portidn ofwhich was then distilled to remove any water. A

small portion {of insoluble materialwas removed bylfilat forty degreescentigrade. Phosgene was then reintroduced into the solution until atotal of about 100 grams had been added. The mixture was allowed tostand for about- 45 minutes and then approximately one half of thesolvent was distilled to remove any dissolved gases.

To the resulting solution of soysteryl chloroformate was added eightygrams of a-naphthylamine in 500 milliliters of dry benzene. The mixturewas refluxed for fifty minutes and eight milliliters of concentratedhydrochloric acid was added to remove the unreacted a-naphthylamine. Theresulting precipitate of 52.4 grams of a-naphthylamine hydrochloride wasfiltered and the filter cake washed well with hot benzene. The benzenewas then distilled from the combined filtrate and washed to leave 141grams, the theoretical yield, of soysteryl a-naphthylcarbamate.

Recrystallization of the thus-produced soysteryl unaphthylcarbamate fromabout 750 milliliters of cyclohexane gave 118.5 grams, a yield of 84percent of the theoretical, of soysteryl a-naphthylcarbamate melting at168 to 185 degrees centigrade and consisting of twenty percentstigmasteryl a-naphthylcarbamate.

F. Soysteryl a-naphthylcarbamate.-ne hundred grams of soysterolscontaining eighteen percent stigmasterol was dissolved in 800 milliliersof cyclohexane and about fifty milliliters of the solvent distilled toremove any water. The hot solution was mixed with five grams of Darcodecolorizing charcoal and filtered through a bed of Celite diatomaceousearth. To the filtrate was added 47.5 grams of a-naphthylisocyanate andfive milliliters of dry pyridine. The solution was refluxed for fivehours and then cooled. The thus-produced precipitate of soysteryla-naphthylcarbamate was filtered and washed with about 100 millilitersof cyclohexane. After drying, the crystals of soysteryla-naphthylcarbamate weighed 118.5 grams, a yield of 82.5 percent of thetheoretical, and melted at 168 to 185 degrees centigrade (Kofier).

G. soysteryl phenylcarbamate.Twenty grams (0.048 mole) of soysterolscontaining eighteen percent stigmasterol was dissolved in 300milliliters of boiling cyclohexane. The solution was slurried with threegrams of Darco decolorizing charcoal and filtered through a bed ofCelite diatomaceous earth. The filtrate was concentrated to 225milliliters by distillation and 6.55 grams (0.055 mole) ofphenylisocyanate and a few drops of pyridine were added thereto. Thesolution was heated at the refluxing temperature of the cyclohexane forfive hours and then allowed to cool to room temperature. The crystalswhich precipitated were filtered, washed with cyclohexane and dried. Thesoysteryl phenylcarbamate thus-obtained weighed 20.3 grams, a yield of79 percent calculated on the soysterols, and melted at 168 to 176degrees centigrade (Kofier).

H. Soysteryl o-biphenylcarbamate.-Following the procedure described inPreparation G, soysteryl a-biphenylcarbamate was prepared in 77 percentyield from soysterols and o-biphenylisocyanate in boiling cyclohexane.The

reaction product crystallized overnight from the cooled cyclohexane andmelted at 178 to 181 degrees centigrade.

I. Soysteryl p-nitrophenylcarbamate.-Following the procedure describedin Preparation G, soysteryl p-nitrophenylcarbamate was prepared in 73percent yield from soysterols and p-nitrophenylisocyanate in methylenechloride. The reaction product crystallized from the concentratedmethylene chloride containing added ethyl acetate anddmethanol andmelted at 217 to 220 degrees centigra e.

I. soysteryl o-t0lylcarbamate.-In the same manner as described inPreparation G, soysteryl o-tolylcarbamate was prepared from soysterolsand o-tolylisocyanate in 63 percent yield and precipitated from thereaction mixture upon distillation of some of the cyclohexanc.

K. Soysteryl p-tolylcarbamate.-In the same manner as described inPreparation G, soysteryl p-tolylcarbamate was prepared in 55 percentyield from p-tolylisocyanate and soysterols and melted at 184 to 187degrees centigrade.

TABLE IV.SINGLE RECRYSTALLIZATIONS OF I 2.5 GRAM SAMPLES OF SOYSTERYLa-NAPH- THYLCARBAMATE Percent Stig- Solvent Solvent Preeipltate masterylRatio Weight a-Naphthylcarbamate 1'? 8t? 28 11 01 62 61 2.0 0.53 63,63 1. 25 0.80 57 Carbon tetrachloride i 5 g: g 2.0 0.38 50, 60 1. 25 0.78 22 Propylene chloride 8: g? 62 2.0 0. 32 61, 62 1'2 82% 3 Ethylacetate 75 1 36 64 2.0 O. 30 62 l? 81% 21 Benzene 1f 75 01 5a 62 2.00.46 60 1. 25 0. 77 57 Chlorobenzene 1: g 33 2.0 0.37 67 Nitrobenzene 1. 25 0. 79 57 Ethyl ether 1. 25 1. 03 49 Methyl ethylketone. 1. 25 0.82 67 Cyolohexane 1. 25 0. 84 48 Methyleyclohexan 1. 250.71 48 Ohloroform 1. 25 0. 48 67 Example 1A.-Fractional crystallizationfrom benzene In the manner described by Morton in Laboratory Techniquein Organic Chemistry, McGraw-Hill Book Company, page 162 (1938), 116.4grams of soysteryl a-naphthylcarbamate containing 21.4 percentstigmasteryl anaphthylcarbamate was crystallized in four stages usingbenzene as a solvent. The first, second and the recrystallized thirdfractions yielded a total of twenty grams of stigmasteryla-naphthylcarbarnate of 87 percent purity, representing a seventypercent recovery of the stigmasteryl a-naphthylcarbamate theoreticallyobtainable. The first fraction consisted of nine grams of stigmasteryla-naphthylcarbamate of 92.3 percent purity, melting at 213 to 218degrees centigrade and having an [@1 in chloroform of minus nineteendegrees. In comparison, stigmasteryl a-naphthylcarbamate prepared fromstigmasterol and a-naphthylisocyanate, after recrystallization from amixture of cyclohexane and methylene chloride, melted at 213 to 217degrees centigrade and had an [M (chloroform) of minus 22 degrees andthe analysis as follows:

Calculated for C H NO C, 82.56; H, 9.53. Found: C, 82.21; H, 9.38.

Sitosteryl a-naphthylcarbamate free stigmasteryl a-naphthylcarbamate,which melted at 172 to 176 degrees centigrade, had an [11],; inchloroform of minus eight degrees, was obtained in the fourth fraction.In comparison, sitosteryl a-naphthylcarbamate prepared by reacting thesitosterols from soysterols with a-naphthylisocyanate melts at about to176 degrees centigrade.

stages into five fractions of thefollowing .13 Example 1B.-,-Fractionalcrystallization from cyclohexane Twenty grams of soysteryla-naphthylc'arba'mate consisting of twenty percent. stigmasteryl.wnaphthylcarbarnate was fractionally crystallized in three stages fromcyclohexane according to the method of Morton, Labora- 'tory Techniquein Organic ChemistryfMc'Graw Hill Book Company, page 162 (1938) togivein the first fraction, 2.71 grams of stigmasteryl a-naphthylcarbamate of82 percentpurity, melting at 204 to 211 degrees centigrade and having an[a] .i11 chloroform of minus seventeen degrees. This yield represents'a"55.5'percentr-'-recovery of the stigmasteryla-naphthylcarbamatepresent in the original mixture.

Fraction three yielded 10.31 grams, a 64.5 percent recovery of thetheoretical amount, of substantially pure TABLE?V Percent Stigmasteryl-Garbw No. of .n1ate O'ontent .Soysteryl Carbamate :Solvent 1 :Stages .F;F2 1F; IFa F175 Fu cyclohexane 4 54 42 24 ethyl acetate -L'; 3 52' "21 4acetone 3 29 27 11 cyclohexane. 4 21 46 30 acetone 5 9 26 CHaCOCzH- 3 4419 14 benzene-.. ,3. 22 13 6 ."benzene;ethnnol, "3 "6 "9 15 ethylacetate.

sitosteryl wnaphthylcarbamate melting at 170 to 174 degrees centigradeand having an [only inchloroform of minus seven degrees.

Example 2.-Crystallization 0f soysteryl a-naphthylcarbamate .Five gramsof soysteryl a-naphthylcarbamate .of' the same composition as that usedin Example l was dissolved in 1.1 liters of Skellysolve C (hexanehydrocarbonsyand two crystal crops collected. The first1.19,.-gramxcrystal .crop contained 49.8 percent stigmasteryl"::anaphthylcarbamate.

The second 0.92-gram crystalxcropwcontained 37.8 percent stigmasteryloc-naphthylcarbamate. The residual mother liquor contained 2.72 vzgrams.of zsolids consisting of sitosteryl a-naphthylcarbamate substantiallyfree of stigmasteryl a-naphthylcarbamate.

Example 3.Crystallization of soysteryl ot-naphthylcarbamate Ten grams ofsoysteryl u-naphthylcarbamate ofithe same composition as that employedin Example 1 was crystallized three times from benzene. The first motherliquor of eighty milliliters contained72 percent of the total solids andcontained 63 percent stigmasteryl a-naphthylcarbamate. The second motherliquor'offortymilliliters contained ten percent of the total solids and''contained 18.4 percent stigmasteryl oz-naphthylcarbamate. The thirdmother liquor contained 0.35 percent of the total solids and contained26.2 percent 'stigmasteryl a-naphthylcarbamate. The crystal croprepresented 14.1 percent of the total solids and contained 86.1 percentstigmasteryl m-naphthylcarbamate.

Example 4.Triangular crystallization 'of soysteryl phenylcarbamateFollowing the crystallization technique shown in Figure 3 of thedrawings [see Morton, Laboratory Techniques in Organic Chemistry,McGraw-Hill (1938), page 162], soysteryl phenylcarbamate of nineteenpercent stigmasteryl carbamate content was separated in fourstigmasteryl phenylcarbamate content:

-Iri Examples 10 and" l 3,-the' car-bamatewhichwas purified was thesitosteryl ca"'rba'xna'vte, which L was increased in purity-from'=a-=starting-=puiity 5f abO11t' 82 perce'tit to a purity 'ofabout-"percent in one of ,the "fractions iii-each example.

'ExampleiMw-iLeaching of soysteryl a-naphthy'lcarbarnate Ten-gramportions of soysteryl a-naphthylcarbamate containing twenty percentstigmasteryl a-naphthylcarbamate were leached with various solvents fromthree to six times, using "equal "portions of solvent 'for' eachleaching. Theiundissolvedi residue was assayed for stigmasteryl oc-"naphthylcarbamate. "Themesultsare shown in Table VI below.

TABLE VI.--SERIAL EXTRACTION OF SOYSTERXL a-NAPHTHYLCARBAMATE WITHVARIOUS-SOL- VENTS Total Nnmf Residue Percent '.I..P. Vol. 01' her fWeight. Stigmas- (C.) Solvent Solvent Leaoh- {Percent teryl aof '(ML)ings Yield) "Naplitbyl- Residue carbamate ether 650 3 14. 4 82 204-213cyclohexane (at,63?)-. 400, 3 11,1 -85 208-215 Skellysnlve O (at 73) 4003 I5. 8 77 204-212 benzene 6 12. 3 88 210-216 -2.5';'gram' s'amples ofsoysteryl a naphthylcarbamate containingapproximately twenty" percentstigmasteryl unapthylcarbamate were leached three times withivarioussolvents. Theratioof the total'volurne of .solventem- [ployedftolthe.calculated amountwas varied with some of the solvents. 'Thecalculatedamount is thefamount necessary ,to dissolve all of the -sitosteryl.mnaphthylcar- .bamate. QThe resultsmflthese experiments are shown inTable VII below. l

TABLE VII TABLE IX Total Volume Percent Weight Percent Yield of WeightStigmasteryl Fraction in stigmasteryl Stigmasteryl of a-Naphthyl- Gramsa-Naphthyla-Naphthyl- Milli- Ratio Residue carbamate carbamate carbamateliters Calcd.

1.70 "-21 1; 1% 15.1 chlorobenzene n6 1'75 .56 77 3.29 20.2 2. .48 785.81 as 2 a a 2-22 ethylacetam 140 1. '75 I59 69 3155 160 2.00 .58 731.81 as "a benzene 26:6 1. '75 I62 72 30.4 2.0 .55 75 gig '2 g}, Example18.-Countercurrent leaching of soysteryl apropylene chloride M8 1.75 75naphthylcarbamate b tt 111 d 1 2g '1' car on e rac ori e l toluene 191.25 0.66 55 141 grams of soysteryl a-naphthylcarbamate contamg$ f gg-gg g? 0 ing eighteen percent stigmasteryl ot-naphthylcarbamate celohexanilnn 167 1:25 1: 29 40 was divided into five 28.2-gram portions.The first pormethyleyclohexane 250 1. l. 19 43 chloroform" 12 L25 0.5076 tlonwas extracted for thirty minutes each with six fifty pyridine.--"18.6 1.25 0. 84 88 milliliter portions of chlorobenzene. The motherliquors from the first two extracts were combined and the sol- Example16.--Leaching of soysteryl u-naphthylcarbamate A 12.5-grani sample ofsoysteryl m-naphthylcarbamate containing eighteen percent stigmasteryla-naphthylcarbamate and the remainder essentially sitosteryla-naphthylcarbamate was leached with six 25-milliliter portions ofchlorobenzene for thirty minutes per portion. The solids in each motherliquor were weighed and analyzed and the solids remaining after eachleaching were dried, weighed and analyzed for stigmasteryla-naphthylcarbamate. The results are shown in Table VIII below.

Example 17.C'0untercurrent leaching of soysteryl anaphthylcarbamateSoysteryl a-naphthylcarbamate containing 21 percent stigmasteryla-napthylcarbamate Was extracted in the fashion shown in Figure 4. A12.5-gramportion (M 'was leached with six 25-milliliter portions ofchlorobenzene .(S to S The residue (R was analyzed for stigmasteryla-naphthylcarbamate. A second 12.5-gram portion (M was leached with thesix mother liquors obtained from the leachings of the first portion, inthe same order. The residue (R was analyzed. A third 12.5-gram portion(M was leached with the six mother liquors obtained from the leachingsof the second portion; in the-same order. The residue (R and the motherliquors (E to E were analyzed. The results are shown in Table IX below.m 4 1 vent removed to leave a distillation residue consisting ofsitosteryl ot-naphthylcarbamate of approximately 98 percent purity. Thelast four mother liquors were combined, the solvent removed and theresidue added to the second 28.2-gram lot. The residue from the sixextractions weighed 3.14 grams.

The second 28.2-gram portion containing the residues from the last fourmother liquors of the extractions of the first portion was extracted inthe same manner with six fifty-milliliter portions of chlorobenzene andthe residues from the last four mother liquors were added to the third28.2-gram portions. The third, fourth and fifth 28.2-gram portions weretreated in the identical manner. From the second, third, fourth andfifth portions was obtained 4.35, 4.48, 4.52 and 4.19 grams,respectively, of insoluble residue from the extractions. The fiveinsoluble residues were combined to give 20.68 grams of stigmasteryla-naphthylcarbamate of approximately 91 percent purity. Additionalstigmasteryl a-naphthylcarbamate of approximately the same purity can beobtained by reworking the last four mother liquors from the extractionof the fifth portion.

The thus-obtained stigmasteryl a-naphthylcarbamate was hydrolyzed with10.4 grams of potassium hydroxide in 45 milliliters of water and 600milliliters of methoxyethanol for two hours at the refluxing temperautreto yield 13.5 grams of stigmasteryl of 91.3 percent purity, melting at167 to 170 degrees centigrade.

Example I9.-. Semiconlinu0us countercurrent leaching of soysteryla-naphthylcarbamate Soysteryl a-naphthylcarbamate consisting of eighteenpercent stigmasteryl a-naphthylcarbamate was leached according to theprocedure shown in Figure 5 of the drawings. A 12.5-gram portion (M wasleached with six 25-milliliter portions of chlorobenzene (S to S leavingan insoluble residue R The last four mother liquors were used, in thesame order, to leach a second 12.5-gram portion (M of the same soysteryla-naphthylcarbamates. The insoluble residue was then leached with twomore 25-milliliter portions of chlorobenzene (8' and 8' The sameprocedure was employed on a third 12.5-gram portion (M of soysterylu-naphthylcarbarnate, using the last four mother liquors from the secondseries of leachings and lastly, two portions of chlorobenzene (S and SThe first two mother liquors (E and E E' and E and E" and E" from eachof the series of leachings con- -three residues and last six extractswere analyzed.

sisted essentially of sitosteryl wnaphthylcarbamate. The The results areshown in Table X below.

TABLE X Percent Stig- Percent Stig- Weight masteryl amasteryl o1-Fractlon Grams Naphthylcar- Naphthylcarbamate bamate RecoveredSimila-rly, other techniques of separation, i. e., sublimation,distillation at high vacuum, chromatography over carbon, Florisilmagnesium silicate, aluminum oxide, or the like, countercurrentextraction and other separation techniques produce more desirableresults when applied to the separation of the mixtures of soysteryla-naphthylcarbamates than to the mixture of soysterols themselves ortheir acetates.

I-IYDROLYSIS OF STIGMASTERYL a-NAPHTHYL- CARBAMATE A. To a solution of2.4 grams (0.06 mole) of sodium hydroxide dissolved in 2.4 millilitersof water and eighty milliliters of Methyl Cellosolve brand ofmethoxyethanol was added twenty milliliters of toluene following by11.64 grams (0.02 mole) of stigmasteryl a-naphthylcarbamate. Theresulting mixture was heated at its refluxing temperature for threehours after which forty milliliters of toluene and a mixture of eightymilliliters of water and twenty milliliters of Methyl Cellosolve wereadded. The mixture was kept at 65 to 75 degrees centigrade whileseparating the aqueous phase which was then extracted with thirtymilliliters of toluene. The toluene phase and toluene extract werecombined and extracted at 65 to seventy degrees centigrade with 100milliliters of 2.0 N hydrochloric acid and then twice with100-milliliter portions of water. The toluene phase was separated anddistilled to dryness to yield 8.0 grams, a yield of 95 percent of thetheoretical, of stigmasterol of 92.2 percent P y ([]n=- Distillation ofthe aqueous phase and extracts gave four milliliters of awater-toluene-Cellosolve ternary boiling at 82 to 89 degrees centigrade;46 milliliters of a water- Cellosolve binary boiling at 99 to 100degrees centigrade and consisting of 15.3 percent Methyl Cellosolve byWeight; eleven milliliters of a water-Cellosolve codistillate boiling at100 to 122 degrees centigrade and sixty milliliters of Methyl Cellosolveboiling at 122 to 123.5 degrees centigrade.

B. Following the procedure described above, but substituting sitosteryla-naphthylcarbamate for the stigmasteryl a-naphthylcarbamate,substantially pure sitosterol is obtained in about 95 percent yield fromthe hydrolysis.

C. To a solution of 10.4 grams of 85 percent potassium hydroxide in 45milliliters of water was added 600 milliliters of Methyl Cellosolvefollowed by 22 grams of stigmasteryl a-naphthylcarbamate. The resultingmixture was boiled for about two hours at its refluxing temperatureuntil a clear solution was obtained. After cooling the solution to roomtemperature there was added 140 milliliters of water with swirling. Theprecipitated stigmasterol was filtered and washed by reslurrying withtwo ISO-milliliter portions of water. After drying at about fiftydegrees centigrade under a vacuum of fifty milliliters of mercury, theprecipitate weighed fourteen grams, melted at 166 to 168 degreescentigrade and assayed 93 percent stigmasterol.

D. Following the procedure described above, the steryl carbamatesdisclosed in the Preparations and the stigmasteryl carbamates shown inTable III were hydrolyzed using water, Methyl Cellosolve and potassiumhydroxide with the exception of the n-butylcarbamates which required 18boiling for two hours in propylene glycol containing potassium hydroxideand a small amount of water, and the diethylcarbamate which requiresboiling for several hours in trimethylene glycol containing water andpotassium hydroxide.

Example 20.Is0lati0n of stigmasterol from soysterols A. Formation ofsoysteryl a-naphthylcarbamate.A 28.2-gram (0.0684-mole) portion ofsoysterols containing about eighteen percent stigmasterol was heated atto degrees centigrade at reduced pressure until the melt ceased tobubble. Dry nitrogen was introduced into the reaction flask and to themelt was added one milliliter of dry 2,4-lutidine and ten milliliters(11.77 grams; 0.0695 mole) of a-naphthylisocyanate. The temperature ofthe mixture rose to to degrees centigrade during the addition and thistemperature was maintained for 35 minutes. The 41 grams of melt waspoured into a crystallization dish in a thin layer to crystallize. Aftercrystallization was complete, the material was broken into smallparticles for the separation of the stigmasteryl a-naphthylcarbamate.

B. Separation of stigmasteryl a-naphthylcarbamate.-A 28.2-gram sample ofthe 41 grams of crude soysteryl or-naphthylcarbamate was extracted withsix fifty-milliliter portions of chlorobenzene, stirring the mixture forabout ten minutes during each extraction. The residue after the sixthextraction weighed 3.17 grams, a 57 percent yield of the stigmasterolpresent in the starting sterols.

C. Hydrolysis 0] separated stigmasteryl a-naphthylcarbamate.-Thethus-obtained 3.17 grams of stigmasteryl a-naphthylcarbamate washyrolyzed in the manner described in the foregoing section entitledHydrolysis of stigmasteryl a-naphthylcarbamate, subparagraph C thereof,in 97 percent yield to give stigmasterol of 91 percent purity.

Example 21.-Is0lati0n of stigmasterol from soysterols A. Formation ofsoysteryl a-naphthylcarbamate.ln exactly the same manner described inExample 20A, 100 grams of soysterols of eighteen percent stigmasterolcontent were converted into 141 grams of soysteryl a-naphthylcarbamate.The yield in this reaction was 100 percent of the theoretical.

B. Separation of stigmasteryl a-naphthylcarbamate.

'Of the 141 grams of soysteryl a-naphthylcarbamate obtained in themanner described above, 50.0 grams were divided into four 12.5-gramsamples. The stigmasteryl a-naphthylcarbamate theoretically present was2.25 grams per sample or a total of 9.0 grams.

Another 12.5-gram sample of this soysteryl a-naphthylcarbamate wasextracted with six twenty-milliliter portions of chlorobenzene, keepingthe mother liquors separate, leaving 1.28 grams of stigmasteryla-naphthylcarbamate. The mother liquors therefore contained a total of11.22 grams of soysteryl a-naphthylcarbamates of 10.8 percentstigmasteryl a-naphthylcarbamate content. The first two of these motherliquors contained sitosteryl a-naphthylcarbamate substantially free fromstigmasteryl a-naphthylcarbamate.

The first of the four 12.5-gram samples described above was extractedsuccessively with the last four mother liquors in the order obtainedfrom the extraction described above and then with two twenty-milliliterportions of fresh chlorobenzene. There remained after these leachings2.16 grams of stigmasteryl a-naphthylcarbamate of about ninety percentpurity. The second, third, and fourth 12.5-gram samples were treated inthe same manner, i. e., extracted with the mother liquors obtained fromthe previous extraction and then with two twenty-milliliter portions offresh chlorobenzene. There was thus obtained 2.26 grams of stigmasteryla-naphthylcarbamate of high purity from the second sample, 2.25 gramsfrom the third and 2.32 grams from the fourth, or a total of 9.09 gramsof stigmasteryl a-naphthylcarbamate of between ninety and 95 percentpurity. The theoretical stigmasteryl a.-naphthylcarbamate content of thefour samples C. Hydrolysis of stigmasteryl nqphthy lcarbamate.

' four so'lid residues obtained from the leaching's described above werehydrolyzed separately to produce four samples of stigmasterol. The firstsample gave 1.39 grams of stigmasterol of ninety percent purity, a 91percent yield; the second, 1.47 grams of 93 percent purity, a 91.4percent yield; the third, 1.46 grams of ninety percent purity, a 91.8percent yield; and the fourth, 1.51 grains of 92 percent purity, a 92percent yield. The .total yield of stigmasterol, of an average 91percent purity, was 5.83 grams of'pure stigmasterol. The theoreticalyield of stigmasterol from these four samples calculated from thestigmasterol content of the starting sterols was 6.4 grams. The'ire- Aprocess for the purification of a steroidwhich comprises the steps of(1) converting a sterol mixture f consisting essentially of stigmasteroland at least one of the sitosterols into a mixture of one of theircorresponding carbamates, (2) fractionati'ng the mixture into adissolved portion and an undissolved portion by contacting the mixturewith organic solvent, (3) separating the portion enriched with thedesired carbamate from the organic solvent and fromthe portion enrichedwith the undesired carbamate, (4) fractionating the portion enrichedwith the desired ca'rbamate into a dissolved portion and an undissolvedportion by'contacting the enriched portion with organic solvent, and (5)repeating steps 3 and 4 at least once.

- 2. The process of claim 1 wherein the fractionation of the mixturewith organic solvent is achieved by leaching the mixture with thesolvent.

,3. The process of claim 1 wherein the fractionation ofi the mixturewith organic solvent .is achieved by crystallizing the mixture with thesolvent. I i J.

4. The process of claim 1 wherein the starting sterol .mixture isconverted to an arylcarbamate.

- 5. Theprocess of claim 1 wherein the starting ste'rol 'mixture isconverted to its a-naphthylcarbamate.

' .6. The process ofclaim 1 wherein the starting sterol mixture isconverted to its phenylcarbamate.

. 7. A process for the separation of a steroidimixture whichcomprisesthe steps of (l) converting a mixture of stigmasterol andsitosterol 'into a mixture of their .correspondingoc-naphthylcarbamates, ,(2) leaching ,the mixture with an organicsolyentthereby fractionati'ng the mixture into an undissolved portion enrichedwith stigmasteryl a-naphthylcarbamate and a dissolved portion enrichedwith sitosteryl a-naphthylcarbamate, (3) separating the undissolvedportion from the organic solvent containing the dissolved portion, (4)leaching the separated undissolved portion enriched withstigmasteryl.a-naphthylcarbamate with an organic solvent therebyfractionating the mixture into an undissolved portion further enrichedwith stigmasteryl e-n'aphthylcarbamate and a dissolved portion, and (5)continuing steps 3 and 4 at least once more and until stigmasteryla-naphthylcarba'mate of desired purity is obtained. 1

8. The process of claim 7 wherein the leaching is conducted at aboutroom temperature.

9. The process of claim 7 wherein the leaching solvent is chlorobenzene.

10. A process for the separation of stigmasteryl'enaphthylcarbamate froma mixture of soysteryl a-riaphthylcarbamates which includes the steps of(1) leaching a portion of stigmasteryl a-naphthylcar bamate'in admixecover'y of the stigmasterol present in the starting so ystero'lsequivalents will be apparent to one skilled in the art and theinventionis therefore to be limited only by the scope of theappendedclaims. g

successive portions of chlorobenzene, wherein each portion of thechlorobenzene has a volume of between about one and about threemilliliters per gram of the original admixture and contains dissolvedstigmasteryl a-naphthylcarbamate and sitosteryl ot-naphthylcarbamate,the amount of sitosteryl a-naphthylcarbamate decreasing and theproportion of stigmasteryl a-naphthylcarbamate to sitosteryla-naphthylcarbamate increasing with successive portions, (2) leachingthe residual undissolved solids with at least one portion ofchlorobenzene which has a volume of between about one and about threemilliliters per gram of the original admixture, (3) separating theundissolved stigmasteryl a-naphthylcarbamate, and '(4) repeating steps1, 2 and 3 employing a second portion of stigmasteryla-naphthylcarbamate in admixture with sitosteryl a-naphthylcarbamate,and employing as the portions of chlorobenzene containing dissolvedstigmasteryl a-naphthylcarbamate and sitosteryl u-naphthylcarbamate, atleast all except the first two of the mother liquors of step 1 in theorder obtained.

11. The process of claim 10 wherein the leaching temperature is aboutroom temperature.

12. A process for the separation of stigmasteryl anaphthylcarbamate'"from admixture with sitosteryl anaphthylcarbamate which includes thesteps of (1) leaching a portion of stigmasteryl a-naphthylcarbamate inadmixture with sitosteryl a-naphthylcarbamate with at least foursuccessive portions of chlorobenzene, wherein each portion of thechlorobenzene has a volume of between about one and about threemilliliters per gram of original admixture and contains dissolvedstigmasteryl int-naphthylcarbamate and sitosteryl a-naphthylcarbamate,the amount of sitosteryl a-naphthylcarbamate decreasing and theproportion of stigmasteryl a-naphthylcarbamate to sitosteryla-naphthylcarbamate increasing with successive portions, (2) leachingthe residual undissolved solids with two successive portions ofchlorobenzene each of which has a volume of between about one and aboutthree milliliters per gram of original admixture, (3) separating theundissolved stigmasteryl a-naphthylcarbamate, and (4) repeating steps,1, 2 and 3 employing a second portion'of stigmasteryla-naphthylcarbamate in admixture with sitosteryl a-naphthylcarbamate andemploying as the portions of chlorobenzene containing stigmasterylanaphthylcarbamate and sitosteryl a-naphthylcarbamate all except thefirst two of the mother liquors of step 1 in the order obtained.

13. The process of claim 12 wherein the leaching temperature is aboutroom temperature and each portion of leaching solvent has a volume ofabout two milliliters per gram of original admixture. I

14. The process which comprises converting a mixture of stigmasterol andsitosterol to a mixture'of stigmasteryl a-naphthylcarbamate andsitosteryl a-naphthy lcarbam'ate and separating at least a'portion ofthe thus-produced stigmasteryl a-naphthylcarbamate and sitosteryla-naphthylcarbamate from each other.

15. The process which comprises reacting a mixture of stigmasterol andsitosterol with a member of the group consisting of (1) a-naphthylisocyanate and (2) phosgene and a-naphthylamine, to produce amixture of stigmasteryl a-naphthylcarbamate and sitosteryla-naplithylcarbamate and separating at least a portion of thethus-produced stigmasteryl -naphthylcarbamate and sitosteryla-naphthylcarbamate from each other.

16. The process which comprisesreacting a mixture of stigmasterol andsitosterol with a-naphthylisocyanate to produce a mixture ofstigmasteryl a-naphthylcarbamate and sitosteryl a-naphthylcarbamate andseparating stigmasteryl a-naphthylcarbamate and sitosteryla-naphthylcarbamate from each other.

.17..-The'process which comprises reacting amixture of stigmasterol andsitosterolwithphosgene followed by'l fdnaphthylamine to produce amixture of stigmasteryl'iture with sitosteryl a-naphthylcarbamate'withat Ieasttwo naphthylcarbamate and t QI'Y PhQfliQbiiQifi? 21 separatingstigmasteryl u-naphthylcarbamate and sitosteryl a-naphthylcarbamate fromeach other.

18. The process which comprises converting the soysterols of thesoysterol fraction of soybean oil to a mixture of soysteryla-naphthylcarbamates and separating stigmasteryl a-naphthylcarbamatefrom the mixture.

19. The process of claim 18 wherein the mixture of soysterylu-naphthylcarbamates is produced by the T630- tion of the soysterolswith a-naphthylisocyanate.

20. The process of claim 18 wherein the mixture of of soysterylo't-naphthylcarbamates is produced by the reaction of the soysterolswith phosgene followed by anaphthylamine.

21. A process for the isolation of sitosterol from admixture withstigmasterol which comprises converting the mixture of stigmasterol andsitosterol to a mixture of stigmasteryl a-naphthylcarbamate andsitosteryl a-naphthylcarbamate, separating the stigmasterylanaphthylcarbamate and sitosteryl a-naphthyl'carbamate, and hydrolyzingthe separated sitosteryl a-naphthylcarbamate to produce sitosterol.

22. A process for the isolation of stigmasterol from admixture withsitosterol which comprises converting the mixture of stigmasterol andsitosterol to a mixture of stigmasteryl a-naphthylcarbamate andsitosteryl a-naphthylcarbamate, separating the stigmasterylu-naphthylcarbamate and sitosteryl ot-naphthylcarbamate, and hydrolyzingthe separated stigmasteryl a-naphthylcarbamate to produce stigmasterol.

23. The process of claim 22 wherein the mixture of stigmasteryla-naphthylcarbamate and sitosteryl m-naphthylcarbamate is produced bythe reaction of the mixture of stigmasterol and sitosterol witha-naphthylisocyanate.

24. The process of claim 22 wherein the mixture of stigmasteryla-naphthylcarbamate and sitosteryl a-naphthylcarbamate is produced bythe reaction of the mixture of stigmasterol and sitosterol with phosgenefollowed by a-naphthylamine.

25. The process of claim 22 wherein the separation is by fractionalsolution in an organic solvent.

26. The process of claim 22 wherein the separation is by leaching withan organic solvent.

27. The process of claim 22 wherein the stigmasteryl a-naphthylcarbamateis hydrolyzed with an alkaline hydrolyzing agent.

28. The process of claim 22 wherein the stigmasteryl a-naphthylcarbamateis hydrolyzed with an alkali-metal hydroxide.

29. A process for the isolation of stigmasterol from a mixture ofsoysterols which comprises heating a mixture of the soysterols andot-naphthylisocyanate between about References Cited in the file of thispatent UNITED STATES PATENTS 2,173,629 Milas Sept. 19, 1939 2,362,932Rosenberg Nov. 14, 1944 2,394,615 Jenkins Feb. 12, 1946 2,503,385 GouldApr. 11, 1950 2,598,468 Vaterrodt May 28, 1952 OTHER REFERENCES Blohm:Chem. Rev. 51, 471-504 (1952). Shriner et al.: Identification of OrganicCompounds, 3rd ed., pp. 159-164, 226-228 (1948).

1. A PROCESS FOR THE PURIFICATION OF A STEROID WHICH COMPRISES THE STEPSOF (1) CONVERTING A STEROL MIXTURE CONSISTING ESSENTIALLY OFSTIGMASTEROL AND AT LEAST ONE OF THE SITOSTEROLS INTO A MIXTURE ONE OFTHEIR CORRESPONDING CARBAMATES, (2) FRACTIONATING THE MIXTURE INTO ADISSOLVED PORTION AND AN UNDISSOLVED PORTION BY CONTACTING THE MIXTUREWITH ORGANIC SOLVENT, (3) SEPARATING THE PORTION ENRICHED WITH THEDESIRED CARBAMATE FROM THE ORGANIC SOLVENT AND FROM THE PORTION ENRICHEDWITH THE UNDESIRED CARBAMATE, (4) FRACTIONATING THE PORTION ENRICHEDWITH THE DESIRED CARBAMATE INTO A DISSOLVED PORTION AND AN UNDISSOLVEDPORTION BY CONTACTING THE ENRICHED PORTION WITH ORGANIC SOLVENT, AND (5)REPEATING STEPS 3 AND 4 AT LEAST ONCE.